I am trying to identify infiltrating NKT cells in mouse mammary tumours. I am not sure whether I still need to incubate cells with α-Galactosylceramide before detection with an anti-Mouse alpha GalCer:CD1d Complex antibody, or whether I can go straight ahead and use the antibody in my cell suspension to pick up already activated NKT cells. The reagents I was thinking of using are http://www.ebioscience.com/mouse-galcer-cd1d-complex-antibody-pe-l363.htm and http://www.abcam.com/alpha-galactosylceramide-ab144262.html.
Thanks Younes but I don't think this is quite what I am looking for. All this chapter says is that you identify them based on CD3 and NK1.1, whereas not all NKT cells are NK1.1+ (see reference). I'd like the most robust way of identifying the cells as possible, and I think using GalCer is now the best and most acceptable way to do this.
http://www.pnas.org/content/105/32/11287.short
Staining with tetramer specific for iNKT is the best way. I am working on that also.
Lucky!
Hi Sarah,
if I understand it correctly, the antibody from eBioscience you suggested will only stain cells expressing CD1d loaded with a-Gal-Cer, so antigen presenting cells, but not NKT cells. For human NKT cells, there is the 6B11 clone directed against the human NKT cell TCR. However, in case of mouse NKT cells, you need to stain them with a loaded CD1d tetramer. We used to request it from the NIH tetramer core facility:
http://tetramer.yerkes.emory.edu/reagents/request-process
In your case, I would ask for a mCD1d tetramer, loaded with PBS-57 (similar to a-Gal-Cer, but with increased solubility). Those coupled to PE, APC and Alexa647 worked best in my hands.
Cheers
Christoph
Thanks for this feedback. I do have access to a small amount of tetramer I can test, so I'll go ahead and use that. It would be great to be able to use the antibody as it is relatively inexpensive, but I won't worry about purchasing that now. Thanks everybody again!
An NIH grant is not necessary to order tetramers from the core facility.
http://tetramer.yerkes.emory.edu/support/faq
You just have to send them a small summary of what you plan to do with the tetramers and to sign an MTA. They will ship the reagent worldwide. You only need to pay for the shipping, the tetramer is for free. You will also receive an unloaded control tetramer.
I've had tetramers form NIH before sent to the UK with no funding from them.
Hi Christoph vahl,
Can you please share your protocol for CD1d staing of mouse splenocytes.
Thank you in advance
Sourav
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